Leveraging the 3-Chloro-4-fluorophenyl Motif to Identify Inhibitors of Tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is implicated in melanin production in various organisms. There is a growing body of evidence suggesting that the overproduction of melanin might be related to several skin pigmentation disorders as well as neurodegenerative processes in Parkinson’s disease. Based on this consideration, the development of tyrosinase inhibitors represents a new challenge to identify new agents in pharmaceutical and cosmetic applications. With the goal of identifying tyrosinase inhibitors from a synthetic source, we employed a cheap and facile preliminary assay using tyrosinase from Agaricus bisporus (AbTYR). We have previously demonstrated that the 4-fluorobenzyl moiety might be effective in interactions with the catalytic site of AbTYR; moreover, the additional chlorine atom exerted beneficial effects in enhancing inhibitory activity. Therefore, we planned the synthesis of new small compounds in which we incorporated the 3-chloro-4-fluorophenyl fragment into distinct chemotypes that revealed the ability to establish profitable contact with the AbTYR catalytic site. Our results confirmed that the presence of this fragment is an important structural feature to improve the AbTYR inhibition in these new chemotypes as well. Furthermore, docking analysis supported the best activity of the selected studied compounds, possessing higher potency when compared with reference compounds.


Introduction
Tyrosinase (TYR, EC 1.14.18.1) is a binuclear copper containing protein expressed in various species including bacteria, fungi, plants, and animals. The main function of TYR is related to its ability to catalyze the oxidation of L-tyrosine and/or L-DOPA to furnish dopaquinone as a melanin precursor in melanosomes. Melanin plays the relevant role of a photoprotectant and is responsible for hair, skin, and eye color. However, an overproduction of melanin is considered to be related to skin disorders. Moreover, the melanin accumulation in human neurons in the substantia nigra is associated with neurodegeneration in Parkinson's disease. The melanogenesis is a very complex and multistep process; however, the TYR activity controls the rate-limiting step, thus determining its pivotal role in the whole process of the synthesis of the physiological pigment. To date, there is a large collection of anti-melanogenic agents from synthetic [1][2][3][4] and natural sources [5][6][7][8][9]. They have been generally identified through the determination of inhibitory properties preventing the oxidation of physiological substrates (L-tyrosine or L-DOPA) in in vitro assays using enzyme from mushroom Agaricus bisporus (AbTYR) as a surrogate to perform a preliminary screening of potential TYR inhibitors (TYRIs). Despite there being several differences between human and mushroom isoforms, the employment of AbTYR enables a facile screening of new potential TYRIs. Interestingly, some hit compounds identified through AbTYR also demonstrated the ability to inhibit the human tyrosinase (hTYR) and produce antimelanogenic effects in human cell lines, although they proved to be less potent agents [10][11][12]. The catalytic cycle of TYR involves two distinct pathways: the hydroxylation of monophenol (monophenolase activity) and the subsequent oxidation of o-diphenol to o-quinone (diphenolase activity). From a structural point of view, the TYRs contain a catalytic pocket in which both monophenolase and diphenolase activity occurs, assisted by the two divalent copper ions surrounded by three cooperating histidine residues [13]. The various classes of reversible TYRIs exert their effects through a network of interactions with copper ions and/or specific residues paving the walls of the catalytic cavity. X-ray determination and in silico studies contributed to the elucidation of the various mechanisms of TYR inhibition as well as to the in-depth analysis of the differences between AbTYR and hTYR.
As a continuation of our previous investigations into AbTYR inhibitors, we planned the synthesis of a new small series of compounds, designed while keeping in mind that the 4-fluorobenzyl motif exerted optimized biological activity, that is, diphenolase activity of AbTYR, owing to its ability to occupy the catalytic cavity, engaging both π/π stacking and the fluorine bonding interaction [14][15][16][17][18]. In addition, it was demonstrated that an additional chlorine atom at the C-3 position of the phenyl ring provided an improvement in AbTYR inhibitory effects [19]. On the basis of these observations, we considered the 3-chloro-4-fluorophenyl moiety as an essential portion to decorate other scaffolds already distinguished as being able to engage favorable contacts within the catalytic cavity of TYRs. Herein, we report the synthesis, in vitro assay, and structure affinity relationship (SAR) considerations for this class of new compounds possessing 3-chloro-4-fluorophenyl in place of the 4-fluorobenzyl pharmacophoric feature. For selected compounds, docking simulations suggested the most plausible binding pose within the catalytic site of TYRs.

In Vitro Assay Determination of AbTYR Inhibition
Our optimization strategy led to the design of sixteen new derivatives (Table 1). To assess the inhibition of AbTYR of new compounds, we employed the method previously described for parent compounds 1a, 2a, 3a, and 4a, with a few modifications. Table 1 col lects the IC50 values of inhibitory effects for the new designed compounds 1d-f, 2c-d, 3b 3d, and 4d-f bearing the 3-chloro-4-fluorophenyl fragment, in comparison with their par ent compounds 1a-c [15,17,18], 2a [20], 3a [14], and 4a [16], which have already been re ported, as well as new analogue derivatives 2b, 3c, 4b-c, 4g, and 4h, which were synthe sized for comparison purposes. All data were compared to the activity of kojic acid (KA) which is considered the canonical reference compound in this screening protocol [15].

In Vitro Assay Determination of AbTYR Inhibition
Our optimization strategy led to the design of sixteen new derivatives (Table 1). To assess the inhibition of AbTYR of new compounds, we employed the method previously described for parent compounds 1a, 2a, 3a, and 4a, with a few modifications. Table 1 collects the IC 50 values of inhibitory effects for the new designed compounds 1d-f, 2c-d, 3b, 3d, and 4d-f bearing the 3-chloro-4-fluorophenyl fragment, in comparison with their parent compounds 1a-c [15,17,18], 2a [20], 3a [14], and 4a [16], which have already been reported, as well as new analogue derivatives 2b, 3c, 4b-c, 4g, and 4h, which were synthesized for comparison purposes. All data were compared to the activity of kojic acid (KA), which is considered the canonical reference compound in this screening protocol [15].

In Vitro Assay Determination of AbTYR Inhibition
Our optimization strategy led to the design of sixteen new derivatives (Table 1). To assess the inhibition of AbTYR of new compounds, we employed the method previously described for parent compounds 1a, 2a, 3a, and 4a, with a few modifications. Table 1 collects the IC50 values of inhibitory effects for the new designed compounds 1d-f, 2c-d, 3b, 3d, and 4d-f bearing the 3-chloro-4-fluorophenyl fragment, in comparison with their parent compounds 1a-c [15,17,18], 2a [20], 3a [14], and 4a [16], which have already been reported, as well as new analogue derivatives 2b, 3c, 4b-c, 4g, and 4h, which were synthesized for comparison purposes. All data were compared to the activity of kojic acid (KA), which is considered the canonical reference compound in this screening protocol [15].  [18]. d Data taken from [15]. e Data taken from [17]. f Data taken from [20]. g Data taken from [14]. h Data taken from [16].
The new 3-chloro-4-fluorophenyl-based benzamide compounds 1d-f achieved significant IC 50 values, spanning from 0.19 to 1.72 µM, as AbTYR inhibitors, showing a light improvement in potency with respect to previous reports for active 4-fluorophenyl-based analogues 1a-c. Based on the confirmation of the activity of compounds containing the 3-chloro-4-fluorophenyl moiety, we further adopted this strategy toward 4-hydroxyphenylbased compounds and studied new analogues of prototype 2a (IC 50 value of 4.49 µM). Considering that the piperazine core could be more prone to being variously decorated, our investigation began with the evaluation of AbTYR inhibitory effects of analogue compound 2b, which was demonstrated to still be an active inhibitor (IC 50 value of 4.43 µM). Subsequently, we introduced the additional 3-chloro-4-fluorosubstituent to furnish the active compound 2c (IC 50 value of 1.73 µM); when a methyl group was added to the acetyl linking group, we obtained the homologue compound 2d (IC 50 value of 1.38 µM), which displayed a similar potency to compound 2c, suggesting that the methyl group occupied a large area of the pocket and that the change in steric hindrance of the linking group did not affect the binding interaction. The chlorine atom was also introduced on the 3-position of the phenyl ring of the previously reported 5-(pyridin-4-yl)-3-(alkylsulfanyl)-4H-1,2,4-triazol-4-aminebased derivative 3a; inhibitor 3b was obtained, which was about 3.5-fold more potent than parent compound 3a. In contrast, the inhibitory effects dramatically disappeared when the chlorine atom was positioned at the 2-position of the phenyl ring, as evidenced for derivative 3c. To gain further information, we replaced the pyridin-4-yl-substituent with the phenyl ring to furnish compound 3d; as a result, the AbTYR inhibitory activity was lower than that of parent compound 3b. Finally, we extended our investigations toward a series of alkylsulfanyl-based compounds 4b-h; these new potential AbTYR inhibitors were inspired by prototype 4a, which emerged from an in silico screening campaign in our previous studies. In the current investigation, we introduced a few structural modifications and replaced the aromatic feature with small alkyl groups. Among the series of compounds 4b-f bearing two aromatic tails, it might be observed that the presence of the 3-chloro-4-fluorophenyl fragment led to a remarkable enhancement in the inhibitory activity; the unsubstituted compounds 4b-c were inactive agents, whereas the poly-substituted derivatives 4d-f displayed inhibitory effects in the low micromolar range (IC 50 values Int. J. Mol. Sci. 2023, 24, 7944 5 of 15 from 2.96 to 10.65 µM); they were still more potent than the precursor 4a. By comparing the IC 50 values of the two methylsulfanyl-derivatives 4g and 4h, we confirmed that the 4-fluorine atom substitution gained better AbTYR affinity than that of the 3-fluorine atom substitution.

Docking Studies on Mushroom Tyrosinase
To confirm the important role of the 3-chloro-4-fluorophenyl fragment in the binding to the AbTYR catalytic pocket, a semi-flexible docking protocol was applied for selected representative compounds 1d, 2c, 3b, and 4f using the software Gold [21]. The results of our molecular modelling analysis of the best potent inhibitors 1d, 2c, 3b, and 4f are depicted in Figure 2. For all of the docked inhibitors, the fluorine atom formed relevant contacts in the AbTYR cavity: (i) it was involved in halogen bond interactions with the copper-coordinating histidine; (ii) it generally acted as a metal acceptor toward di-copper ions CuA and CuB, with the exception of the compound 3b, which interacted exclusively with CuA (see Figure 2C); and (iii) it formed hydrophobic contacts with Phe292. Furthermore, we observed that the 4-fluorosubstituted phenyl ring interacted by π-π stacks with the side chain of His263. Particularly, in compounds 1d, 3b, and 4f, the different fragments characterizing each chemotype showed a similar rearrangement, engaging favorable contacts with the residues forming the entrance of the catalytic site, such as Val248, Met257, Asn260, and Phe264. A distinguishable binding mode is observed for compound 2c, probably due to its greater length compared with the other candidates. As a result, folding of the 4-piperazine-phenyl tail occurs to accommodate the ligand in the pocket, stabilized by hydrophobic contacts with the side chain of His244 and Val283 and a hydrogen bond between the phenolic OH group and Cys83, supporting that the increase in chain length should not A distinguishable binding mode is observed for compound 2c, probably due to its greater length compared with the other candidates. As a result, folding of the 4-piperazinephenyl tail occurs to accommodate the ligand in the pocket, stabilized by hydrophobic contacts with the side chain of His244 and Val283 and a hydrogen bond between the phenolic OH group and Cys83, supporting that the increase in chain length should not affect the binding affinity.
Moreover, despite that a different fitting of 3-chloro-4-fluorophenyl is encountered in all compounds, the chlorine atom stabilized the binding to the catalytic cavity through van der Waals interactions, specifically with Ala286 and Val283 in the ligand 1d and with Phe90 and Val283 in the inhibitors 2c, 3b, and 4f. The collected results corroborated the importance of this moiety and were consistent with the biochemical data displayed in Table 1.

Chemistry
The synthesis of the designed compounds was performed employing the synthetic routes described in Schemes 1-3. The preparation of the designed compounds followed the procedures described in our previous publications [14,18,20], with slight modifications. Scheme 2. Synthesis of the designed compounds 3b-d [14]. Reagents and conditions: (i) NaOH, MeOH, rt, 3 h, quantitative.
The first series of three target compounds 1d-f was readily prepared in three steps in a moderate yield via the N-alkylation reaction, removal of the protective group, and condensation with carboxylic acid or acyl-chlorides, as reported in Scheme 1. In detail, the key The synthesis of the compounds 3b-d bearing the 4H-1,2,4-triazol-4-amine core was carried out by the coupling of reactants 10a-b with the suitable benzylbromide 11a-b under basic conditions, as displayed in Scheme 2.
All compounds were structurally characterized through spectroscopic measurements, as reported in Section 3 and the Supplementary Materials.

Biochemical Assays
Mushroom tyrosinase (EC 1.14.18.1) was supplied by Merck (Cat. No. T3824). In vitro assay was performed according to the method of Mirabile et al. [15]. Each sample (0.05 mL) at different concentrations (0.10-200 µM) was mixed with 0.5 mL of L-DOPA solution (1.25 mM) and 0.9 mL of phosphate buffer solution (pH 6.8). After a 10 min incubation at 25 • C, 0.05 mL of an aqueous solution of the enzyme was added to the mixture. Absorbance was recorded at 475 nm using a spectrophotometer (Agilent Technologies, Cary 60, UV/Vis). Kojic acid [5-hydroxy-2-(hydroxymethyl)-4-pyran-4-one] was employed as a positive standard (10-30 µM). The inhibition rate was calculated with the following mathematical equation: where A = absorbance of negative control and B = absorbance of test sample. Finally, IC 50 values were determined by interpolation of the dose-response curves.

Molecular Modelling
Docking studies were carried out using Gold software V 2020.2.0 [21]. To achieve this purpose, our studies were carried out on the basis of the structure of the complex of Agaricus bisporus tyrosinase (AbTYR) and tropolone (PDB ID 2Y9X) [25]. The protocols applied for the protein and ligands' preparation and the docking calculation have been reported in our previous works [18,20]. The analysis of the docked poses was carried out by means Discovery Studio Visualizer [26] and Ligand Scout V 4.4.9 [27].